In recent years, a great deal of information has been accumulated on the molecular alterations that take place during the development of tumors, such as gene mutations or genomic rearrangements, highlighting the possibility of detecting tumor alterations in biological fluids and consequently indicating the use of these markers as a valid non invasive diagnostic approach.
A tumor that has been widely investigated with this approach is colorectal cancer, which is one of the most common forms of cancer worldwide, with a clinical outcome varying considerably according to the type of lesion and stage of disease at diagnosis (1-3). An early diagnosis is fundamental to reduce morbidity and mortality as a high percentage of patients diagnosed in the early stages of disease are long-term survivors (4). Moreover, the possibility of detecting pre-malignant lesions makes this tumor an ideal target for screening programs. However, although several screening methods are available, a high percentage of individuals do not participate in colorectal cancer screening programs. There are many reasons for this low compliance, such as a lack of knowledge of the benefits of the available screening methods, especially colonoscopy, as well as the unpleasant and troublesome procedures (5).
Gene mutations in stool, especially K-ras (6-12) and to a lesser extent p53 (13), APC gene (14,15) and microsatellite instability (16), have been repeatedly investigated. Results have shown the presence of these molecular alterations in stool in only a fraction of patients, due to the relatively low frequency of single marker alterations in colorectal cancer. Multiple mutations have been analyzed in parallel on the same stool sample and this approach has led to improved test sensitivity, but is expensive, time-consuming and cannot easily be applied to screening programs (17-21).
The diagnostic potential of DNA amplification of exfoliated cells in stool has recently been considered. Preliminary evidence (19-21) has shown that the semi-quantitative evaluation of DNA amplification (L-DNA) of some DNA fragments longer than 200 bp detects more than 50% of colorectal cancers, with a very high specificity.
US application No. 20020004206 discloses a genetic assay for identifying a tumor disease from samples containing exfoliated epithelial cells. The patent application describes an assay comprising a step of PCR amplification of Kras, APC and p53 fragments, followed by semi-quantitative determination of the amplified DNA based on gel-staining.